Preparation of phosphatides

ABSTRACT

Modified phosphatides of improved emulsifying power and suitable for use as anti-spattering agents in margarine are obtained by the partial hydrolysis of a vegetable phosphatide with an enzyme preparation containing lipase and phospholipase A until at least 2 percent and less than 15 percent of lysophosphatides are formed, and removal of free fatty acids by solvent extraction.

United States Patent Pardun [54] PREPARATION OF PHOSPHATIDES [72]Inventor: Hermann Pardun, Cleves, Germany [73] Assignee: Lever BrothersCompany, New York,

[22] Filed: Jan. 8, 1970 [2]] Appl. No.: 1,559

[30] Foreign Application Priority Data Jan. 9, 1969 Germany ..P 19 00959.7

[52] U.S. Cl ..l95/30, 99/15, 99/123, 260/403 [51] Int. Cl. ..Cl2b 1/00[58] Field of Search ..195/2, 3, 12, 30; 99/15, 17, 99/50, 60, 90;206/403; 252/1 [56] References Cited UNITED STATES PATENTS 3,576,8313/1971 Davis ..260/403 [451 Mar. 28, 1972 3,047,597 7/1962 Pardun..99/l5 UX OTHER PUBLICATIONS Unilever, N. V.; Chem. Abs., Vol. 70, No.79434W, 1969. Wittcofi, H.; The Phosphatides, p. 99- 1 15, 1951.Oliefabrik, A.; Chem. Abs., Vol. 63, No. 13590 A, 1963. Unilever, N. V.;Chem. Abs., Vol. 70, No. l 14,012f, 1969.

Primary Examiner-A. Louis Monacell Assistant Examiner-Gary M. NathAttorney-Louis F. Kline, Jr., Melvin H. Kurtz and Edgar E. Ruff 57ABSTRACT Modified phosphatides of improved emulsifying power andsuitable for use as anti-spattering agents in margarine are obtained bythe partial hydrolysis of a vegetable phosphatide with an enzymepreparation containing lipase and phospholipase A until at least 2percent and less than 15 percent of lysophosphatides are formed, andremoval of free fatty acids by solvent extraction.

6 Claims, No Drawings PREPARATION OF PHOSPHATIDES This invention relatesto processes for the preparation of partially hydrolyzed vegetablephosphatides.

Crude hydrated vegetable phosphatides (or plant lecithins) are obtainedas by-products in the solvent-extraction of oilseeds for edible oilproduction. In typical commercial process (as more fully described inBritish Pat. No. 1,118,373) they are precipitated as slimes by theintroduction of moist steam into the solvent-free crude vegetable oil,and then separated from the bulk of the oil by centrifugation and driedby evaporation under reduced pressure. The dried crude phosphatidesconsist of a mixture of about 65 percent by weight of materialsinsoluble in acetone, namely choline,

ethanolamine, serine and inositol phosphatides, sugars and glycolopids,and about 35 percent of acetone-soluble substances, mainly neutral oil,free fatty acids and unsaponifiable constituents.

Such vegetable phosphatides are used as emulsifying agents forwater-in-oil emulsions, for example margarine, and also for oil-in-wateremulsions, for example mayonnaise and salad oils,

but the choline phosphatide components on the one hand and v theethanolamine and serine phosphatide components (together known ascephalin) on the other, antagonise or depress the emulsifying power ofeach other.

Danish Pat. No. 101,649 describes the improvement of the emulsifyingpower of crude vegetable phosphatides by treating aqueous oil-containingvegetable phosphatide emulsions with lipase at from 50 to 80 C. forseveral hours. The lipase effects a partial hydrolysis of the fatty acidtriglyceride of the oil to a mixture of monoand diglycerides, giving aproduct of improved emulsifying power, but an undesirable increase infree fatty acid content occurs, as a result of which the taste of theproducts thus prepared is less pleasant than that of normal vegetablephosphatides. Phosphatides can be converted into lysophosphatides byhydrolysis with the enzyme phospholipase A (lecithinase A) occurringtogether with lipase in pancreas extract or pancreatin, or by pancreatinwhich has been partially heat-inactivated to give a preparation havingonly phospholipase A activity.

It has now been found that a modified phosphatide emulsifying agent ofneutral taste and light color and suitable for oilin-water andwater-in-oil emulsions can be prepared by the partial hydrolysis of anunhydrolysed vegetable phosphatide with an enzyme preparation containingboth lipase and phospholipase A at from to 70 C. until there has beenformed at least 2 percent and less than 15 percent of' lysophosphatides(monoacyl glycerophosphatides) by weight of the total phosphatides andlysophosphatides present, followed by separation of these by solventextraction from free fatty acids present.

Suitable unhydrolysed vegetable phosphatides for use in the process arethe crude hydrated vegetable phosphatides referred to above, forinstance hydrated soyabean, groundnut, sunflower, corn and rapeseed oilphosphatides. Such starting materials can be the crude phosphatideslimes which have been separated from the bulk of the oil, for instanceby centrifugation, and such a slime that has been subsequently dried,and such a slime which with or without subsequent drying has beensubjected to further purification by extraction of the fatty acidtriglyceride oil it contains by fat solvents, for example acetone. Therecan also be used phosphatide fractions obtained by solvent fractionationof the crude hydrated vegetable phosphatides, for examplealcohol-soluble and alcohol-insoluble fractions, especiallyalcohol-soluble fractions with a proportion of phosphatidylcholine tophosphatidylethanolamine of at least 4 to l by weight, such as aredescribed in British Pat. No. 1,1 13,241.

Suitable enzyme preparations containing lipase and phospholipase A aretechnical enzyme preparations of plant 'or animal origin, particularlytechnical extracts from pancreas,'such as pancreatin. Theenzymepreparations can be conveniently used in amounts of from 0.05 to0.2 percent, preferably 0.05 to 0.1 percent, based on the weight ofphosphatide employed.

The partial hydrolysis is conveniently carried out in the presence of atleast 25 percent, and preferably at least 50 percent of water, by weightof the phosphatide to be hydrolyzed, and preferably at from 40 to 50 C.

The rate of hydrolysis increases with the amount of enzyme preparationemployed, so that the reaction time required for a particular degree ofhydrolysis is reduced. Typical reaction times are from 3 to 60 hours.Preferably hydrolysis is carried out until a lysophosphatide content offrom 4 to 12 percent is reached.

The crude partially hydrolysed vegetable phosphatide (or hydrolysate)obtained is then subjected to extraction with a solvent suitable forseparation of free fatty acids from the phosphatides andlysophosphatides present. The hydrolysate can be dried beforeextraction, but preferably the direct product of hydrolysis (containingthe water present during hydrolysis) is used for the extraction.

Preferably the hydrolysate is dispersed in an organic solvent medium inwhich the free fatty acids are soluble and the phosphatides andlysophosphatides are insoluble, and a partially hydrolyzed phosphatideis then recovered from the insoluble residue.

The hydrolysate can be dried and dispersed in a first organic solvent inwhich the free fatty acids, phosphatides and lysophosphatides dissolve,a second organic solvent in which the free fatty acids are soluble butthe phosphatides and lysophosphatides are insoluble then added to thesolution until a phase containing the latter is precipitated, and theprecipitated phase separated and purified partially hydrolysedphosphatide recovered from it. For the first solvent are suitablealiphatic, cycloaliphatic and aromatic hydrocarbons of from five toseven carbon atoms, for example pentane, hexane, heptane, cyclohexane,methylcyclohexane, and benzene, and chlorinated hydrocarbons of one ortwo carbon atoms, for example dichloromethane, dichloroethane,trichloroethylene and carbon tetrachloride. There can for example beused from 0.1 to 1.5 liters of hexane per kg. hydrolysate calculated asfree of water. For the second solvent are suitable ketones or alkylesters of carboxylic acids containing from two to four carbon atoms, forexample acetone, methyl acetate and ethyl formate. There can for examplebe used from 1.5 to 3 liters of acetone per kg. dried hydrolysate. Theextraction and precipitation can be repeated. The product after additionof both solvents forms two liquid phases, of which one phase containsoil, fatty acids and other undesired materials, and the other phasecontains the phosphatides and lysophosphatides. After separation of thephases, the phase containing the phosphatides and lysophosphatides isfreed from solvent, for example by evaporation under reduced pressure.

Preferably the hydrolysate is dispersed in an alcohol or ketone of fromone to three carbonatoms, for example methyl, ethyl, propyl or isopropylalcohol or acetone, and water is present: aqueous solventsconsisting of2 parts by volume of alcohol or ketone to from 1 to 3 parts of water canbe used. The partially hydrolysed phosphatide is preferably firstthoroughly stirred with a little alcohol or ketone, and the aqueousalcohol or ketone then added until at least two layers are formed.Usually three are formed of which the uppermost consists of dark-coloredneutral oil and fatty acids, the middle one consists of a solution ofbitter substances and sugars in aqueous solvent and the lowermostconsists essentially of phosphatides, lysophosphatides and solvent. Forthe separation of these layers a centrifuge is convenient. Aqueousacetone containing from 5 to 30 percent of water can also be used, inwhich case two layers may be formed of which the upper layer containsthe oil and the fatty acids and the lower layer contains thephosphatides and lysophosphatides. Where the hydrolysate alreadycontains a sufiicient amount of water, the alcohols or ketones can beadded free of water.

Where sufficient water is present a chlorinated hydrocarbon solvent suchas one of those mentioned above can be used as the sole solvent; theproportions of solvent and water should be such that the free fattyacids are in one phase and the phosphatides and lysophosphatides in theother.

After the phase containing the phosphatides and lysophosphatides hasbeen separated, it is freed from solvent, for example by evaporationunder reduced pressure. As the purified partially hydrolyzed vegetablephosphatide is liable to deteriorate in air by oxidation, carriersubstances, for example neutral fatty acid triglyceride oils, mixturesof fatty acid monoand diglycerides, glycerol, sorbitol,propane-1,3-diol, and ethyl lactate, can be added before the finalevaporation process to avoid this.

The products are emulsifying agents which can be used in the preparationof foodstuffs, for example margarine, mayonnaise and salad oils,feedingstuffs, for example synthetic calf milk, cosmetic preparations,for example lotions and salves, and pharmaceutical preparations.

The invention is illustrated by the following Examples, in whichtemperatures are in C.

EXAMPLE 1 A crude hydrated soyabean phosphatide slime (1,000 g.) wasstirred with water (100 g.) until an emulsion was formed, technicalpancreatin (l g.) added and the emulsion heated for 16 hours at 50.After removal of water by evaporation under reduced pressure there wasobtained a crude partially hydrolyzed phosphatide (1,000 g.) of acidnumber 33 and iodine color 40, containing 64 percent acetone-insolubles,15.6 percent choline phosphatide, 8.5 percent cephalin and 3.0 percentlysophosphatide by weight: the amount of lysophosphatide present thuscorresponded to about 4% percent by weight of the total phosphatides andlysophosphatides. Half of this product was dissolved in hexane (500 m1.)and extracted with acetone (l,l ml., containing 2.5 percent water byweight). The upper layer formed on standing was separated off and thelower layer extracted twice with hexane (550 ml.) and acetone (l,l00ml.). To the residue of purified partially hydrolyzed phosphatide wasadded a refined soyabean oil in equal amount to the oil removed in theextraction, and solvent was removed by evaporation under reducedpressure. The purified hydrolysate had an acid number of 19.7 and aniodine color of 25.

For determination of the emulsifying power of the product, water (50ml.) at 50 was placed in a 100 m1. mixing cylinder, a solution of thepurified hydrolysate (1 g.) in groundnut oil (9 g.) of melting point 32was added and a coarse emulsion prepared by rotation of the cylinderthrough 180 times. The cylinder was then placed in a waterbathmaintained at 50 and the time in hours required for separation of ml.water (half-value time) was noted. The test was carried out both withdistilled water and water of 13 hardness, and a comparison was made withthe crude partially hydrolysed phosphatide that had not been subjectedto the extraction process.

Half-value time (hi1) A crude hydrated syoabean phosphatide slime (2,000g.) containing 56.6 percent by weight of dry substance was mixed withtechnical pancreatin (500 mg.) and allowed to stand for 51 hours at 60.The acid number of the mixture on a dry weight basis increased duringthis time from 20 to 38. The crude hydrolysate thus obtained contained50 percent by weight of water.

A portion (400 g.) of the crude hydrolysate was freed from water at 70by evaporation in a rotary evaporator under reduced pressure to give adried crude hydrolysate (201 g).

Another portion (400 g.) of this crude hydrolysate was stirred withacetone (1,000 ml.) at ambient temperature. After long standing theturbid upper layer was poured off from the layer of sediment and thelatter then extracted again with acetone (1,000 ml). 0n evaporation ofthe reduced extract under reduced pressure there was obtained a dark,fatty acidrich oil (70 g). The residue after addition of refinedsoyabean oil (70g) was evaporated under reduced pressure to give apurified hydrolysate (200 g.) (Example 2).

A further portion (400 g.) of the crude hydrolysate was stirred withisopropyl alcohol (200 ml.) for l5 minutes at ambient temperature, andformed three layers on standing. The mixture was then centrifuged toseparate the upper and middle layers from the lower layer. Onevaporation of the combined upper and middle layers there was obtainedan oil (44 g). Refined soyabean oil (44 g) was added to the lower layerand the mixture evaporated under reduced pressure to give a purifiedhydrolysate (20! g.) (Example 3).

A final portion (400 g.) of the crude hydrolysate was stirredintensively at 20 for 30 minutes with l,2-dichloroethane 1,200 ml.). Onstanding there was deposited a lower layer containing phosphatide, fromwhich the oil-containing dichloroethane layer was separated first bydecantation and then by centrifugation, and on evaporation under reducedpressure there was obtained oil (71 g). To the residual phosphatide wasadded refined soyabean oil (71 g.) and the mixture evaporated underreduced pressure to give a purified hydrolysate (201 g.) (Example 4).

The dried crude and purified hydrolysates were subjected to analysis, totesting for taste threshold value, namely the lowest concentration ofthe produce in groundnut oil that could still be detected by half themembers of a tasting panel, and to testing for anit-spattering action inmargarine. For the latter purpose margarines were prepared containingpercent fat, 4 percent milk, 0.2 percent starch, and 0.2 percent salt byweight, and incorporating a series of amounts of the hydrolysates. Themargarines thus obtained were heated by the methods described in Fette,Seifen, Anstrichminel, 1963, 65, 29, and the formation of spatterdetermined on the visually assessed basis of 8 very good 4unsatisfactory 7 good 3 bad 6 satisfactory 2 very bad 5 adequate lunusable The results were as follows:

Purified hydrolysate according to Example Crude hydrolysate The amountof lysophosphatide (5.0 percent) present in the dried crude hydrolysatecorresponds to about 7% percent by weight of the total phosphatides andlysophosphatides.

What is claimed is:

l. A process for the preparation of a modified phosphatide emulsifyingagent which comprises partially hydrolysing an unhydrolysed vegetablephosphatide with an enzyme preparation containing both lipase andphospholipase A at from 30 to 70 C. until at least 2 percent and lessthan percent of lysophosphatides by weight of the total phosphatides andlysophosphatides present is formed, and then separating by solventextraction the phosphatides and lysophosphatides from free fatty acidspresent.

2. A process according to claim 1, in which the unhydrolysed vegetablephosphatide is a crude hydrated vegetable phosphatide slime.

3. A process according to claim 1, comprising dispersing the hydrolysatein an organic solvent medium in which the free fatty acids are solubleand the phosphatides and lysophosphatides are insoluble, and recoveringa partially hydrolysed phosphatide from the insoluble residue.

4. A process according to claim 3, comprising dispersing the driedhydrolysate in a first organic solvent in which the free fatty acids,phosphatides and lysophosphatides dissolve, adding to the solution asecond organic solvent in which the free fatty acids are soluble but thephosphatides and lysophosphatides are insoluble until a phase containingthe latter is precipitated, and separating the precipitated phase andrecovering from it a purified partially hydrolysed phosphat'ide.

5. A process according to claim 3, comprising extracting the directproduct of hydrolysis.

6. A process according to claim 5, in which the organic solvent mediumis an alcohol or ketone of from one to three carbon atoms.

# i t t i

2. A process according to claim 1, in which the unhydrolysed vegetablephosphatide is a crude hydrated vegetable phosphatide slime.
 3. Aprocess according to claim 1, comprising dispersing the hydrolysate inan organic solvent medium in which the free fatty acids are soluble andthe phosphatides and lysophosphatides are insoluble, and recovering apartially hydrolysed phosphatide from the insoluble residue.
 4. Aprocess according to claim 3, comprising dispersing the driedhydrolysate in a first organic solvent in which the free fatty acids,phosphatides and lysophosphatides dissolve, adding to the solution asecond organic solvent in which the free fatty acids are soluble but thephosphatides and lysophosphatides are insoluble until a phase containingthe latter is precipitated, and separating the precipitated phase andrecovering from it a purified partially hydrolysed phosphatide.
 5. Aprocess according to claim 3, comprising extracting the direct productof hydrolysis.
 6. A process according to claim 5, in which the organicsolvent medium is an alcohol or ketone of from one to three carbonatoms.